AU2018266135B2 - Hydroxyurea to enhance sperm cells - Google Patents

Abstract

A method of enhancing sperm fertility is described herein. The method includes contacting sperm with hydroxyurea. A composition is disclosed that includes sperm, hydroxyurea, and a buffer. Methods, kits, and compositions for enhancing sperm fertility are also provided.

Description

HYDROXYUREA TO ENHANCE SPERM CELLS

[0001] This is a PCT Patent Applicaton filed ibr "Hydroxyurea to Enhance Sperm Cells." This application claims

priority to and benefit of co-pending U.S. provisional patent application number 62/503,131, filed M ay 8, 2017.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This inventon was made with government support under grant(s) awarded by the Natonal Institutes of

Health (NIH) Grant number: RO1 HD020419-1951 and IU54HD0431501-09 awarded by U.S. Nabonal Institutes

of Health. The government has certain rights in the invention.

[0003] In this context "government' refers to the government of the United States of America.

BACKGROUND OF THE DISCLOSURE

[0004] Mature sperm cells (spermatozoa) are the male reproductive cells in sexual reproduction in animals,

including humans. A sperm cell is a male gamete br fusion with a female gamete (ovum or egg cell). Aler fusion

of the gametes, the resulting cell, a zygote, normally develops into an embryo.

[0005] Sperm has many characteristics that are important to fertility, including morphology and motility. Sperm

motlity, or the ability of sperm to swim properly to effectuate ferblization, is one such important characteristic of

sperm in determining success of ferblization, both natural and artiicial. In mammals, such as humans, effective

sperm motlity facilitates the passage of sperm through the cumulus oophorus and the zona pellucida, which

surround the female reproductive egg cell, to complete ferblization of the female reproductive cell. In the case of

internal ferblization, sperm motlity is measured by the degree by which sperm cells move through the female

reproductive tract to reach and ferblize an egg cell. In artiicial inseminaon, sperm molity is measured by the

ability of sperm cells to move through an aqueous solution to reach and ferblize an egg cell.

[0006] Human sperm cells have a flat, disc shaped head and a long flagellum, or tail. The tail flagellates, or

whips, to propel normal sperm in a direction opposite of the tail at a rate of about 1-3 mm/minute. The lagellum is

principally responsible br sperm molity.

[0007] Human sperm cells reach full motility (hyperactive molity or hyperactivity) as a result of environmental

pH changes, such as in the uterine lumen afler ejaculated sperm penetrates the ovulatory alkaline cervical mucus.

During natural inseminabon, hyperactvity is typically triggered when the alkaline nature of seminal fluid, the

medium of sperm during male ejaculaton, is neutralized by the acidic vaginal environment. Hyperactvity is

characterized by a non-directonal vigorous moon pattern, which is a characteristic moon pattern of sperm that

have acquired the ability to bind to and ferblize mature eggs.

[0008] Sperm moblity may be measured by a variety of parameters, including by moblity grade. Moblity grade

may be divided into ibur different classiicatons of actve (i.e., non-passive) moblity, which in order of decreasing

moblity, are: Grade a, Grade b, Grade c and Graded, also known as moblity IV, moblity Ill, moblity || and moblity

I, respectvely. Grade a sperm moblity is characterized by sperm that moves quickly and in aforward and straight

direction opposite of the sperm tail. Grade b sperm moblity is characterized by sperm that moves fbrwardly in a

curved or indirect path. Grade c sperm motlity is characterized by sperm that moves generally with tail movement

but fails to move forward along a path. Grade d sperm motlity is characterized by sperm that is immolle.

[0009] When sperm possess low sperm moblity, ferilizabon is less likely to be achieved, as the sperm is not able

to move effectively to reach an egg cell. In the context of assisted reproductive treatments, such as in vitro

ferilizabon (IVF) and intrautrine inseminaton, alow sperm moblity also decreases the likelihood of successful

frtilizabon, as the low sperm moblity results in sperm that are unable to effectvely move through an aqueous

environment to ferblize an egg cell.

[0010] Human sperm cells are environmentally sensitve, and as such, many environmental factors to which the

human sperm cells are exposed may cause low or reduced sperm moblity. Examples of activies in male subjects

that result in such detrimental environmental factors include: tobacco use, marijuana use, alcohol consumpton,

anabolic steroid use, and useof testosterone supplements. Further detrimental factors to sperm moblity in subjects

include excessive stress, old age, excessive heat exposure to genitals, side effects of medicaton, and poor diet.

As a large population of human male subjects have sperm that is affected by one or more of these detrimental

factors, there is a need fr technologies to enhance sperm moblity.

SUMMARY

[0011] It has unexpectedly been discovered that hydroxyurea has positive effects on sperm motlity, longevity

and other factors related to sperm health, including in sperm of humans.

[0012] In a first aspect, a method of enhancing sperm moblity is provided, comprising contacting hydroxyurea to

sperm.

[0013] In a second aspect a compositon ibr enhancing sperm motlity is provided that comprises an effective

amount hydroxyurea and a buffer, such as human tubal fluid medium.

[0014] In a third aspect, a kit for enhancing sperm moblity of sperm is provided. The kit includes hydroxyurea

and a bufler, such as human tubal fluid medium or modified human tubal fluid medium.

[0015] In a iburth aspect a plurality of treated sperm having enhanced moblity are prepared by a process

comprising contacting sperm with an effective amount of hydroxyurea.

[0016] In a fifh aspect a method of suppressing human immunodeiciency virus ("HIV') in semen is provided.

The method includes providing semen fom a subject infected with the HIV and contactng the semen with

hydroxyurea.

[0017] In a sixth aspect a method of preventing HIV transmission fom an HIV positive sperm donor is provided.

The method includes providing semen from the HIV positive sperm donor, contactng the semen with hydroxyurea,

and ferblizing an ovum with the sperm fom the post-contacted semen.

[0018] In a seventh aspect, a method of increasing sperm longevity is provided that comprises providing sperm

and contacting the sperm with an effectve amount of hydroxyurea.

[0019] In an eighth aspect a method of increasing a yield of post-thaw sperm sample after cryopreservabon is

provided. The method includes providing a sample comprising a plurality of spermatozoa, contactng the

spermatozoa with hydroxyurea, subjectng the spermatozoa to cryopreservalon, and thawing the spermatozoa.

[0020] The above presents a simplified summary in order to provide a basic understanding of some aspects of

the claimed subject matter. This summary is not an extensive overview. It is not intended to identfy key or critcal

elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in

a simplified form as a prelude to the more detailed description that is presented later.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGS. 1A-1H depict photographs taken over about 9 seconds showing sperm not processed with

hydroxyurea(control).

[0022] FIGS. 2A-2H depict photographs taken over about 9 seconds showing sperm processed with hydroxyurea

(experimental).

DETAILED DESCRIPTION

[0023] Unless otherwise defined, all terms (including tchnical and scientiic terms) used herein have the same

meaning as commonly undersbod by one of ordinary skill in the art of this disclosure. It will be further understood

that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that

is consistent with their meaning in the context of the specificaton and should not be interpreted in an idealized or

overly frmal sense unless expressly so defined herein. Well-known functions or constructons may not be

described in detail br brevity or clarity.

[0024] The terminology used herein is fr the purpose of describing partcular embodiments only and is not

intended to be limitng. As used herein, the singular brms "a", "an" and "the" are intended to include the plural

fbrms as well, unless the context clearly indicates otherwise.

[0025] The term "consistng essentially of' means that, in addition to the recited elements, what is claimed may

also contain other elements (steps, structures, ingredients, components, etc.) that do not adversely affect the

operability of what is claimed r its intended purpose as stated in this disclosure. This term excludes such other

elements that adversely affect the operability ofwhat is claimed br its intended purpose as stated in this disclosure,

even if such other elements might enhance the operability of what is claimed br some other purpose.

[0026] The terms "about' and "approximately" shall generally mean an acceptable degree of error or variaton

br the quantty measured given the nature or precision of the measurements. Typical, exemplary degrees of error

or variaton are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of

values. For biological systems, the term "about' refers to an acceptable standard deviaton of error, preferably not

more than 2-bld of a given value. Numerical quanties in this detailed description are approximate unless stated

otherwise, meaning that the term "abouf' or "approximately" can be inferred when not expressly stated.

[0027] The terms "individual," "subject," or "patent' as used herein refer to any animal, including mammals, such

as mice, rats, other rodents, rabbits, dogs, cats, swine, catie, sheep, horses, primates, and humans. The terms

may specify male or female or both, or exclude male or female.

[0028] The term "spermatozoon" refers to a live male reproductive cell. The term "spermatozoa" refers to a

plurality of live male reproductive cells. Unless required otherwise by context, the plural and singular ibrms are

interchangeable. The term "sperm"is used as an abbreviabon and refers to at least one spermatozoon. As used

herein, sperm may be from a human male subject or an animal, including domestcated animals, such as bovines,

equines, or swine.

[0029] Hydroxyurea ("HU"), also known as hydroxycarbamide, has a chemical formula of CH4N202 and chemical

structure of

0

HIN ',N' H Surprisingly, it has presently been discovered that, among other things, hydroxyurea enhances the moblity of

sperm and has no adverse effects on sperm at beneficial concentratons. This is surprising as it was previously

believed that HU was detrimental to male and female ferblity.

[0030] A method of enhancing sperm motility may include contactng sperm with hydroxyurea. The sperm may

be sperm that were isolated from semen from a human male or another animal such as bovine. The hydroxyurea

may be contained in a suitable buffer, such as human tubal luid medium or modified human tubal fluid medium,

described further below. The hydroxyurea may be present in the buffer in an amount effectve for enhancing sperm

moblity, such as in an amount of from 0.1 to 250 pg/mL, fom 1 to 50 pg/mL,from 10 to 25 pg/mL, about 18 pg/mL,

or any subvalue and/or subrange thereof. These concentratons may also reflect the concentration of hydroxyurea

in the post-contacted sperm and buffer mixture. The method may increase progressive motility, i.e., linear

movement fom one point to another, of the spermfrom sperm that are otherwise non-progressive, i.e., sperm that

move but do not makeibrward progression.

[0031] In some embodiments, the enhanced moblity comprises activated hyperactve motlity, also known as

hyperachvated sperm mollity. Hyperachvated sperm mollity is characterized by sperm that have a high amplitude,

asymmetrical beating pattern of the flagellum. Hyperachvatd sperm moblity is more vigorous and short term than

progressive motlity. Biologically, hyperachvated sperm moblity is important toenable sperm to traverse the egg

outer investments prior to ferblizing the mature egg. The sperm motlity may be enhanced prior to artiicial

inseminabon of the sperm. The method may include providing semen and isolating sperm fom the semen. The

method includes contactng the sperm with hydroxyurea to process the sperm. The buffer may comprise human

tubal fluid medium or modified human tubal luid medium. In an embodiment the hydroxyurea processed sperm is

incubatd ibr a period of between 1 minute to 24 hours, 15 minutes to 3 hours, 30 minutes to 1.5 hours, about 1

hour, or any subrange and/or subvalue thereof

[0032] As earlier provided, the sperm may be contacted with the hydroxyureain a suitable buffer. Buffer is often

used in procedures that require sperm washing, such as intrauterine inseminabon and in vitro ferblizabon. Sperm

washing removes mucus (seminal plasma) and non-moble sperm fom the semen. Thus, the hydroxyurea

containing buffer serves to simultaneously increase sperm mollity and to wash the sperm, which advantageously

combines a washing buffer and hydroxyurea to increase moblity in a single compositon and step. The buffer may

comprise hydroxyurea in any amount effective to produce a desired result, such as enhancing sperm moblity or

suppressing HIV, ibr example, of from 0.1 to 250 pg/mL, from 1 to 50 pg/mL, from 10 to 25 pg/mL, about 18

pg/mL, or any subrange or subvalue thereof The buffer may be, ibr example, human tubal luid ("HTF) medium

or modified HTF medium. HTF comprises a sodium bicarbonate buffering system and may be utlized fr uses

requiring a carbon dioxide atmosphere during incubaton. Modified HTF comprises acombined sodium bicarbonate

and HEPES ([4-2(2-hydroxyethyl)-1-piperazineethanesulibnic acid]) buffer. Modiied HTF provides maintenance of

physiological pH (7.2 to 7.4) and is also appropriate ibr uses not requiring a carbon dioxide atmosphere during

storage. Suitable examples of HTF medium or modified HTF medium include those that are commercially available

from Irvine Scientic, Santa Ana, Calibrnia. The sperm buffer solution may be incubated ibr a period sufficient to

provide a measurable improvement in the moblity (or other characteristics) of the sperm; in specific embodiments of the method, incubaton is fom 1 minute to 24 hours, 15 minutes to 3 hours, 30 minutes to 1.5 hours, about 1 hour, or any subrange or subvalue thereof

[0033] In an embodiment of the method, a washing step is peribrmed. The washing may include centrifuging

sperm with a mixture of HTF-HEPES medium comprising 0.5% bovine serum albumin ("BSA") and separating and

extractng the centrifuged sperm. The washing step may be peribrmed before, simultaneously with, or after the

sperm is processed with hydroxyurea.

[0034] A method of increasing spermlongevity is provided, comprising providing sperm and contacting the sperm

with an effectve amount of hydroxyurea to process the sperm. The effectve amount of hydroxyurea may befrom

0.1 to 250 pg/mL, fom 1 to 50 pg/mL,from 10 to 25 pg/mL, about 18 pg/mL, or any subrange or subrange thereof.

The effectve amount of hydroxyurea may be up to a therapeutcally safe amount of hydroxyurea. As the oral LD5o

of hydroxyureain a single dose is 5780 mg/kg in rats, dividing by 1000 to calculate a rough maximum safe dose

ibr humans, a maximum effective dose may be 5.78 mg/kg or less. While not impossible, it is unlikely that the

hydroxyurea of the present disclosure would be administered orally. However, the maximum effectve dose of 5.78

mg/kg based on the oral LD5o in rats may be a useful guidepost in determining a maximum safe hydroxyurealevel

in humans, whether administered orally or via another route.

[0035] Without being bound by any partcular hypothesis, it is believed that the increase in motlity caused by

hydroxyurea processing of sperm contribute to a longer longevity in sperm. Thus, sperm processed with

hydroxyurea are able to be stored ibr a longer period of tme and retain the ability to fertlize an egg cell as

compared to sperm not processed with hydroxyurea.

[0036] In some embodiments, a method of increasing a yield of post-thaw sperm after sperm cryopreservaton

includes providing a sample comprising the sperm, contactng the sperm with hydroxyurea, subjecting the sperm

to cryopreservaton, and thawing the sperm. An increased yield of sperm means that a higher relative number of

sperm are mole and capable of ferilizaton as compared to non-HU processed sperm. Thus, sperm processed

with HU have a higher yield after cryopreservaton and thawing than sperm not processed with HU. The

hydroxyurea contacted sperm may also include any of several useful cryopreservatve components, including one

or more components such as glycerol, citrate, egg yolk, andandbacterial agents. Glycerol may be present at any concentration known in the art to preserve cellular viability upon freezing; examples of such concentrations include about 5-25% v/v, more specifically about 10-20% v/v, 12% v/v, and 15% v/v. Citrate and egg yolk can be used to reduce osmobo stress on the cells during penetraton by glycerol. An example of a suitable cryopreservalve component, a freezing bufler, is "Freezing Medium - TYB with Glycerol &Gentamicin," available commercially by

Irvine Scientic of Santa Ana, Caliibrnia, which contains, among other ingredients, 20% egg yolk, 12% glycerol,

and 10 pg/mL gentamicin sulfate. Anibios can be used to reduce the likelihood of microbial contaminaton of

the sample. Such components may be used at concentrations that would be used in standard glycerol and glycerol

egg yolk cryopreservalve. In an embodiment, the hydroxyurea is provided in a feezing bufler.

[0037] The method may include storing the mixture ibr a period of one day to 20 years, 30 days to 5 years, at

least 2 weeks, at least a month, at least a year, or any subrange or subvalue thereof In some embodiments of the

method, the cryopreservabon comprises freezing at a temperature suficienly low for effective cryopreservalon;

ibr example, the cryopreservabon may be conducted at about -10° C or below; in further embodiments the

compositon is fozen at -40° C or below; in sill further embodiments thetemperature is about -80°, -100°, -140°,

-160°, -180°, or -190° C, and below.

[0038] In another aspect, hydroxyurea may be used in the manufacture of a compositon ibr the preventon of

HIV transmission or in the manufacture of a compositon ibr enhancing sperm motility.

[0039] In an embodiment, the hydroxyurea of the present disclosure enhances a motlity grade of sperm. For

example, hydroxyureatreatment mayenhance the sperm motility of sperm having Grade c sperm moblity to Grade

a sperm moblity. Hydroxyurea treatment may enhance sperm moblity of sperm having Grade b sperm motility to

Grade a sperm motility. Hydroxyurea treatment may enhance sperm moblity of sperm having Grade c sperm

moblity to Grade b sperm moblity.

[0040] Advantageously, hydroxyurea maybe used to enhance sperm moblity prior to arbicial inseminabon. As

hydroxyurea acivats, or induces, hyperactve moblity in sperm, hydroxyurea may be used to induce hyperactive

moblity in sperm during an artiicial inseminaton process to increase likelihood of successful ferilization of an egg

cell, such as an egg cell of a human female or a domesticated female animal, such as a female swine or bovine.

[0041] In an embodiment hydroxyurea may be used to activate hyperactve motlity in sperm in an ex vivo

fertlizaton method, such as an in vitro fertlizaton method.

[0042] In another embodiment, a compositon comprises sperm, an effectve amount of hydroxyurea, and a

buffer. The bufler may be any buffer that is suitable as a medium fr viable sperm, such as HTF or modified HTF

medium. The hydroxyureamaybe present in the buffer in an amountoffrom 0.1 to 250 pg/mL, from 1 to 50 pg/mL,

fom 10 to 25 pg/mL, about 18 pg/mL, or any subrange and/or subvalue thereof.

[0043] In some embodiments, akitfr enhancing motlity of sperm comprises an effective amountof hydroxyurea

and a bufler. The buffer may be any buffer disclosed as suitable above. The kit may consist essentially of, or

consist solely of, the effective amount of hydroxyurea and the buffer, such as HTF or modified HTF. The

hydroxyurea maybe provided in the buffer or separately to be mixed later. The hydroxyurea maybe present in the

buffer in any effectve amount, br example from 0.1 to 250 pg/mL, from 1 to 50 pg/mL, from 10to 25 pg/mL, about

18 pg/mL, or any subrange and/or subvalue thereof The hydroxyurea and the buffer may be contained separately,

or together, in a suitable container or containers in the kit.

[0044] In an embodiment a plurality of sperm with signiicanty enhanced motlity are provided that are the

product of a process comprising providing sperm and contactng the sperm with hydroxyurea. The significantly

enhanced motility may be enhanced by at least 10%, at least 20%, at least 50%, or at least 75% or about 80%.

The signiicanty enhanced motlity may be enhanced by from 10% to 200%, fom 25% to 150%, from 50% to

100%, or from 70% to 90%. The signiicanly enhanced motlity may be significant enhanced acivaton of

hyperactve motlity. The signiicanly enhanced motlity refers to Ihe number of sperm cells in an HU treated sample

able to ferlize an egg as compared to those in a non-HU treated sample.

[0045] In another embodiment hydroxyurea may be used in a method to process a semen sample infected with

the human immunodeficiency virus ("HIV'). The method may be an ex vivoprocess, which has the advantages of

more controlled processing conditions and lack of possible side effects in the donor. The processing of the sperm

may comprise suppressing HIV replicaton, expression, and/or acivaton in semen or semen sample. In some

embodiments, the processing prevents HIV transmission between an HIV positive spermatozoon donor and a spermabzoa recipient The recipient may be, fr example, a human female subject having an egg cell inseminated by the sperm of the HIV positive sperm donor.

[0046] Hydroxyurea may be added to a medium containing semen from a subject infected with the HIV. The

hydroxyurea may be added to process the sperm prior to inseminaton of the sperm recipient Hydroxyurea may

be present in the culture medium in any amount effectve to suppress HIV in semen, such as fom 0.1 mM to 10

mM, fom 0.5 mM to 5 mM, from 0.75 mM to 2.5 M M, or 1.0 mM,or any subrange and/or subvalue thereof The

sperm may be incubated in the HU containing medium (i.e., left in contact with the hydroxyurea) br a period

suficient to have a beneicial effect r example, from 1 minute to 24 hours, fom 15 minutes to 3 hours, from 30

minutes to 1.5 hours, about 1 hour, or any subrange and/or subvalue thereof Without being bound by any

partcular hypothetcal model, it is believed that HU depletes deoxyribonucleotde triphosphate ("dNTP") in

simulated peripheral blood lymphocytes ("PBLs"). The depleton of dNTP signiicanly reduces the rate of HIV-1

(the most widespread strain of HIV) DNA synthesis and inhibits the completion of viral DNA synthesis in

phytohemagglutnin (PHA)-stmulated PBLs.

[0047] In an embodiment, hydroxyurea of the present disclosure is used in a method of deactvaing HIV in

semen prior to feezing, or cryopreservabon, of HIV infected semen. In another embodiment, hydroxyurea of the

present disclosure is used in a method of deactvatng HIV in semen during an inferblity treatment In some

embodiments, the hydroxyurea of the present disclosure is used br deactvaing HIV in semen priorto mole sperm

isolaon from semen prior to sperm preparation fr arbicial inseminabon or in vitro ferblizabon.

[0048] In any of the embodiments disclosed herein, the hydroxyurea may be opionally separated, or washed,

from the post-contactd sperm or semen with hydroxyurea. This optonal separating step may be advantageous,

as direct human exposure to hydroxyurea may have harmful side effects. Any suitable means of separating the

hydroxyurea fom the post-contacted sperm may be used, such as column chromatography, iltraton, ordifferenti al

centrifigaton.

[0049] Working Example 1

Human sperm was processed with HU to show enhanced motility over human sperm not processed with HU.

Normal human semen was collected in 15 mL conical centrifuge tubes. Highly mole (i.e., Grade a) sperm were isolated and collected from the semen. The isolated highly mole sperm were washed in an HTF medium (HTF

HEPES; pH 7.4) containing 0.5% Bovine Serum Albumin ("BSA") by centrifugaton and suspended in bicarbonate

(25 mM)-buflered HTF containing 0.5% (5mg/ml) BSA (36; pH 7.4). Subsequenly, each sperm suspension was

incubated at 370C in an atmosphere of 5.0% C02 in humidiied air untl aliquoted into Ihe different treatment groups:

a control group and a hydroxyurea processed group.

[0050] The control group was cultured in HTF-bufler containing 0 pg HU/mL at 370C in an atmosphere of 5.0%

C02 in humidified air fr 24 hours. After the 24 hour culture, the control sperm were evaluated br pattern and

degree of moblity.

[0051] The hydroxyurea processed group was cultured in HTF-bufler containing 18 pg HU/mL at 370C in an

atmosphere of 5.0% C02 in humidified air br 24 hours. After the 24 hour culture, the hydroxyurea processed sperm

were evaluated br pattern and degree of motlity. The resulting overall sperm moblity (progressively mole

spermatozoa [spermatozoa that swim most in a straight line or in very large circles] + hyperacively mole

spermatozoa [spermatozoa that exhibit higher amplitude of lateral head displacement, and lower fequency of

progressive motlity]) as the number of mole spermatozoa in a hundred cells was viewed with a phase contrast

microscope and counted with a laboratory counter. The percentage of mole spermatozoa was calculated by

multplying the radio of mole to 100 spermatozoa counted by 100. The non-hydroxyurea treated group (control)

had an overall percentage moblity value of 75.4 with a standard error of +8.6%, while the hydroxyurea processed

group had a sperm motility of 79.0 with a standard error of +7.0%.

[0052] Measuring, as described above, only the resulting progressive sperm motlity, the control group had a

moblity of 51.7 +10.2%, while the hydroxyurea processed group had a sperm moblity of 37.7 with a standard error

of +8.4%. The progressive moblity of the control group sperm was higher (P<0.001; based on paired t-test

comparison) than the progressive moblity of the hydroxyurea processed group sperm. Surprisingly, measuring only

the resultng hyperactve moblity, as described above, the control group had a hyperactve motlity of 23.0 with a

standard error of +6.4%, while the hydroxyureaprocessed group had a hyperactive motlity of 41.3 with a standard

error of +5.3%. The hydroxyurea processed group possessed signiicanly higher (P<0.001; based on paired t test comparison) hyperactve mole spermatozoa compared to spermatozoa in the control group. These results indicate that hydroxyurea simulates hyperactve moblity in sperm, thus increasing the number of sperm that have acquired the ability to bind to and ferilize mature egg cells. On the other hand, most of the sperm cells in the control buffer remained in a progressive motlity state (moblity pattern observed among sperm cells that have not acquired the ability to ferblize mature eggs). These indings are clinically significant, as progressive sperm moblity is a determinant of ability of spermatozoa to penetrate cervical mucuson towards migrabon to the fertlizabon site

(fallopian tubes), while that fr hyperactive sperm motlity (capacitabon) is the moon pattern characteristic of

spermatozoa that have attained the ability to ferblize mature eggs.

[0053] Working Example 2

Human sperm was processed with HU to show that in the context of preventing HIV transmission, HU has no

significant negative side effects on overall sperm moblity. This result is surprising, as it had been previously

believed that HU was an unsuitable agent to suppress HIV replicaton and/or acivabon in sperm, or to reduce the

risk of HIV transmission via HU treatment of reproductive cells, due to adverse effects of HU on reproductve cells,

such as human sperm and human egg cells, including adverse effects on overall sperm motlity and survival.

[0054] Normal human semen was collected in 15 mLconical centrifuge tubes. Highly mobile (i.e., Grade a) sperm

were isolated and collected from the semen. The isolated highly mole sperm were washed in HTF medium

containing 0.5% BSA (HTF-HEPES; pH 7.4) by centrifugaton and suspended in bicarbonate (25 mM)-buffered

HTF containing 0.5% (5mg/ml) BSA (36; pH 7.4). Subsequenly, each sperm suspension was incubated at 370C

in an atmosphere of 5.0% C02 in humidiied air untl aliquoted into the different treatment groups: a control group

and a hydroxyurea processed group.

[0055] The control group was cultured in HTF-buffer containing 0 pg HU/mL at 370C in an atmosphere of 5.0%

C02 in humidified air fr 24 hours. After the 24 hour culture, the control sperm were evaluated ibr pattern and

degree of moblity.

[0056] The hydroxyurea processed groups were cultured in HTF-buffer in two groups containing different

concentrations of HU: (1) 1.0 mM HU and (2) 2.0 mM HU. The groups were cultured at 370C in an atmosphere of

5.0% C02 in humidified air ibr 24 hours. Alter the 24 hour culture, the hydroxyurea processed sperm were

evaluated ibr pattern and degree of motility.

[0057] Surprisingly, measuring overall moblity, the HU treatEd groups had similar overall moblity measurements

as compared to Ihe control group. The control group had an overall moblity of 70.3 with a standard error of+3.6.

The 1.0 mM HU-reated group had an overall moblity of 66.6 with a standard error of +2.6. The 2.0 mM HUtreated

group had an overall moblity of 68.3 with a standard error of +4.0. These results are surprising, as previously it

was thought that HU was damaging to reproductive cells such as sperm and would reduce sperm moblity. Thus, it

had been previously believed that HU was an unsuitable agent to suppress HIV replicaton and/or achvabon in

semen, or to reduce the risk of HIV transmission, via HU treatment of reproductive cells.

[0058] Working Example 3

Human sperm was processed with HU to show that HU is beneicial for maintaining and preserving spermatozoa

subjected to cryopreservabon. This result was surprising, as HU processing, or contacting of sperm, showed that

hydroxyurea processed sperm is superior ibr maintaining spermatozoa in a feezing medium ibr 24 hours when

compared to sperm that has not been processed or contacted with hydroxyurea.

[0059] Post semen analysis was conducted of discarded and de-identied semen samplesfrom Ovaon Ferility

Center that met the World Health Organizabon (WHO) criteria ibr normal semen samples. Those semen samples

that met the WHO criteria ibr normal were divided into two halves. One half was subjected tofreezing with TEST

Yolk buffer (freezing bufler [FB]; Irvine Scientiic, CA) containing 12% glycerol as cryoprotectant (control group).

The remaining half of each sample was frozen with experimental feezing bufler composed of FB + HU (Sigma

Aldridge, St. Louis; treatment group). Both control and experimental buflers were added on a1:1 radio to respectve

semen samples (v/v) in a dropwise manner. The inal glycerol concentraton in the semen + FB was 6%, and the

final HU concentration in semen + experimental freezing bufler was 1 pM to use a concentraton of HU that is

eflectve in inhibitng HIV replicaton. Pre-freeze sperm moblity was determined ibr each sample priorto initating

the cooling step of the freezing procedure. Samples in the control and treatment group were cooled ibr 24 hours

to slow the HU-induced vigorously mole cells in the treatment group. Subsequenly, post cooling moblity was

determined prior to liquid nitrogen vapor phase freezing ibr 30 minutes beibre being stored immersed in liquid nitrogen (LN) ibr at least 24 hours beibre being thawed for analysis. Samples were thawed at room temperature fbr 35 minutes prior to moblity analysis.

[0060] The results, as shown in Table 1, below, indicate that supplementaton of feezing medium with HU

maintained 76-84% moblity of the initial population of mole spermatozoa (pre-feeze) which is significanly higher

than that observed fbr spermabzoa in the control group (28-45% of pre-freeze population . Furthermore, longevity

was higher among post-thaw spermatozoa fozen in the treatment (HU) group versus those in the control group.

Table 1.

Sample CONTROL CONTROL CONTROL Post- Experimental Experimental Experimental No. Pre-cool Post-cool cool Motility Pre-cool Post-cool Post-cool Motility Motility Motility Relative to Pre- Motility (%) Motility (%) Relative to Pre (%) (%) cool Motility cool Motility (Survival of (Survival of cooling process; cooling process; %) %) 1 47 27 57.4 47 40 85.1 2 62 29 47.8 62 37 59.7 3 44 18 40.9 44 29 65.9 4 63 26 41.3 63 36 57.1 54 22 40.7 54 40 74.1 6 38 29 76.3 38 33 86.8 z 79 40 50.6 79 59 74.7

[0061] As can be seen in Table 1, above, HU is highly effectve at preserving, maintaining moblity in, and

maintaining survival of, mole spermatozoa that are subsequently subjected to cryopreservabon as compared to

non-HU tread mole spermatozoa. Indeed, in every one of the samples tested above, HU treatment increased

yield of mole spermatozoa as compared to non-HU treated mole spermatozoa. The mean of the control post

cool moblity relabve to post-cool moblity (survival of cooling process in percentage) of the seven samples was

ibund to be 50.71 with a standard error of±4.86. Advantageously, the mean of the post-cool moblity relabve to

post cool moblity (survival of cooling process in percentage) of the seven HU treated samples was ibund to be

71.91 with a standard error of ±4.39.

[0062] FIGS. 1A-1H and FIGS. 2A-2H demonstrate the eflectveness of HU at preserving, maintaining moblity

in, and maintaining survival of, mole spermatozoa that are subjected to cryopreservalon. FIGS. 1A-1H illustrate sill screens fom a video showing non-HU treated (i.e., control) mole spermatozoa over about 9 seconds, with

FIG. 1A corresponding to about 1 second elapsed, FIG. 1B corresponding to about 2 seconds elapsed, FIG. 1C

corresponding to about 4 seconds elapsed, FIG. 1D corresponding to about 5 seconds elapsed, FIG. 1E

corresponding to about 6 seconds elapsed, FIG. 1F corresponding to about 7 seconds elapsed, FIG. 1G

corresponding to about 8 seconds elapsed, and FIG. 1H corresponding to about 9 seconds elapsed. As can be

seen, alow level of overall spermatozoa with moblity exists in the control group, with few, if any, spermatozoa

exhibitng hyperactive moblity. On the other hand, FIGS. 2A-2H illustrate sill screens from a video showing HU

treated (i.e., experimental) mole spermatozoa over about 9 seconds, with FIG. 2A corresponding to about 1

second elapsed, FIG. 2B corresponding to about 2 seconds elapsed, FIG. 2C corresponding to about 4 seconds

elapsed, FIG. 2D corresponding to about 5 seconds elapsed, FIG. 2E corresponding to about 6 seconds elapsed,

FIG. 2F corresponding to about 7 seconds elapsed, FIG. 2G corresponding to about 8 seconds elapsed, and FIG.

2H corresponding to about 9 seconds elapsed. As can be seen, a moderate to high level of overall spermabzoa

with moblity exists in the treatment group, with numerous spermatozoa exhibitng hyperactive moblity as compared

to the control group that has low post thaw moblity.

[0063] These results of this Working Experiment 3 are significant, as they demonstrate that HU treatment is

highly effectve in preserving, or maintaining, the viability and moblity of mole spermatozoa that have been

subjected to cryopreservabon. Moreover, HU treatment preserves or maintains the moblity of mole spermatozoa

at HU concentrations that are effective to inhibit HIV replicaton, such as in concentratons of 1 pM, as tested here.

This dual feature (increasing moblity while inhibitng HIV replicaton) of the HU described herein is an important

aspect of the embodiments of this disclosure.

[0064] Working Example 4

Human sperm was processed with HU to show that HU is beneficial br maintaining motility of spermatozoa

subjected to cryopreservabon.

[0065] Post semen analysis was conducted of discarded and de-identied semen samplesfrom Ovabon Ferility

Center that met the World Health Organizabon (WHO) criteria ibr normal semen samples. Those semen samples

that met the WHO criteria ibr normal were divided into two halves. One half subjected to freezing with TEST-Yck buflr(freezing buffer [FB]; Irvine Scientic, CA) containing 12% glycerol as cryoprotectant (control group). The remaining half of each samplewasfrozen with experimental freezing buffer composed of FB+ HU (Sigma Aldridge,

St Louis; treatment group). Both control and experimental buffers were added on a 1:1 radio to respecbve semen

samples (v/v) in a dropwise manner. The final glycerol concentration in the semen +FB was 6% and thefinal HU

concentration in semen + experimental freezing buffer was 1 pM to use a concentration of HU that is effective in

inhibibng HIV replicaton. Pre-feeze sperm motlity was determined ibr each sample prior to initaing the cooling

arm of the freezing procedure. Samples in the control and treatment group were cooled br 24 hours to slow the

HU induced vigorously mole cells in the treatment group. Subsequently, post cooling moblity was determined prior

to liquid nitrogen vapor phase feezing ibr 30 minutes bebre being stored immersed in LN br at least 24 hours

beibre being thawed fbr analysis. Samples were thawed at room temperature fbr 35 minutes prior to moblity

analysis.

[0066] The results, as shown in Table 2, below, indicate that supplementaton of feezing medium with HU

maintained motility of the inital population of mole spermatozoa (pre-freeze) better than the control group, which

did not contain HU.

Table 2.

Sample Pre-cool Post-cool Post-thaw Pre-cool Post-cool Post-thaw No. Motility Motility Motility/ Survival Motility Motility Motility/Survival (Control;%) (Control; Relative to Post- (HU;%) (HU; %) Relative to Post %) cool Motility cool Motility (Control; %) (HU; %) 1 57 56 37/66 57 66 50/76 2 70 34 24/71 70 65 32/49 3 59 49 28/57 59 62 4471

[0067] As can be seen above, HU treatment of mole spermatozoa positvely effects post-thaw moblity in

spermatozoa that have been subjected to cryopreservaton. It should be noted, however, that an experiment

including a higher number of samples than was tested in Working Example 4 would provide br greater stabst cal

accuracy. These results indicate that mole spermatozoa that are contacted with HU have a relatively greater post

thaw moblity and survival rate when compared to the control group of mole spermatozoa.

[0068] PROPHETIC EXAM PLE 5

It is believed that HU treated sperm is safe and effectve ibr ferblizabon of an egg and subsequent zygote

development (e.g., cleavage, compacton, cell division, blastulaton, implementaton, and embryogenesis). An

experiment will be implemented, where samples of eggs, such as eggsfrom swine, bovine, or cricetnae (hamster)

will be artificially ferblized (such as by IVF) with two sets of sperm samples, where one set has been treated with

HU (experimental) and the other set has not been treated with HU (control). For example, if there are 20 samples

of sperm, the 20 samples will be divided into two sets - 20 samples for HU treatment and 20 control samples

without HU treatment, such as by a buffer that does not contain HU. The sperm may be provided from an animal

such as a swine, bovine, or cricetnae. The sets of sperm will then be used to ferblize eggs, preferably eggsfrom

a single animal or a single animal per sample to minimize experimental variables. Subsequent development of the

ferblized eggs of the sets of samples will be observed, measured, and recorded. By comparing development

between the control and HU tread samples, it can be shown that sperm from HU treated semen samples are

safe and effectve ibr animal ferilization procedures and techniques.

[0069] It is to be understood that any given elements of the disclosed embodiments of the inventon may be

embodied in a single structure, a single step, a single substance or the like. Similarly, a given element of the

disclosed embodiment may be embodied in multple structures, steps, substances or the like.

[0070] The ibregoing description illustrates and describes the processes, machines, manufactures, compositons

of matter, and other teachings of the present disclosure. Additonally, the disclosure shows and describes only

certain embodiments of the processes, machines, manufactures, compositons of matter, and other teachings

disclosed, but as mentoned above, it is to be understood that the teachings of the present disclosure are capable

of use in various other combinatons, modiicatons, and environments and are capable of changes or modiicalons

within the scope of the teachings as expressed herein, commensurate with the skill and/or knowledge of a person

having ordinary skill in the relevant art The embodiments described hereinabove are further intended to explain

certain best modes known of practcing the processes, machines, manufactures, compositons of matter, and other

teachings of the present disclosure and to enable others skilled in the art to ulize the teachings of the present

disclosure in such, or other, embodiments and with the various modificatons required by the particular applicatons or uses. Accordingly, the processes, machines, manufactures, compositons of mater, and other teachings of the present disclosure are not intended to limit the exact embodiments and examples disclosed herein. Any section headings herein are provided only ibr consistency with the suggestions of 37 C.F.R. §1.77 or otherwise to provide organizatonal queues. These headings shall not limit or characterize the inventon(s) setibrth herein.

Claims (16)

CLAIMS What is claimed is:

1. A method of enhancing sperm motility, comprising: providing a spermatozoon, and contacting the spermatozoon with hydroxyurea provided in a buffer, wherein the hydroxyurea is present in the buffer in an amount of 10 to 25 pg/mL and the buffer comprises human tubal fluid medium or modified human tubal fluid medium.

2. The method of claim 1, further comprising incubating the contacted spermatozoon for a period of between 1 minute and 24 hours.

3. The method of claim 1, wherein the spermatozoon is human spermatozoon or bovine spermatozoon.

4. The method of claim 1, wherein the enhancing sperm motility comprises activating hyperactive motility.

5. A composition, comprising: a plurality of spermatozoa, and hydroxyurea provided in a buffer to enhance motility of the plurality of spermatozoa, wherein the hydroxyurea is present in the buffer in an amount of 10 to 25 pg/mL and the buffer comprises human tubal fluid medium or modified human tubal fluid medium.

6. A kit for enhancing sperm motility of spermatozoa, consisting essentially of: hydroxyurea for enhancing sperm motility, and a buffer comprising human tubal fluid medium or modified human tubal fluid medium, wherein the hydroxyurea is present in the buffer in an amount of 10 to 25 pg/mL.

7. Treated spermatozoon with enhanced motility prepared by a process comprising the steps of: providing a spermatozoon, and contacting the spermatozoon with hydroxyurea provided in a buffer, wherein the hydroxyurea is present in the buffer in an amount of 10 to 25 pg/mL and the buffer comprises human tubal fluid medium or modified human tubal fluid medium.

8. The treated spermatozoon prepared by the process of claim 7, further comprising incubating the contacted spermatozoon for a period of between 1 minute and 24 hours.

9. The treated spermatozoon prepared by the process of claim 7, wherein the spermatozoon is human spermatozoon or bovine spermatozoon.

10. The treated spermatozoon prepared by the process of claim 7, wherein the enhancing motility comprises activating hyperactive motility.

11. A method of suppressing human immunodeficiency virus activity in semen, comprising: providing semen from a subject infected with the human immunodeficiency virus, and contacting the semen with hydroxyurea provided in a buffer, wherein the buffer comprises human tubal fluid medium or modified human tubal fluid medium and the hydroxyurea is present in the buffer in an amount of 10 to 25 pg/mL.

12. A method of increasing spermatozoon longevity, comprising: providing spermatozoon, and contacting the spermatozoon with hydroxyurea provided in a buffer to increase said longevity, wherein the hydroxyurea is present in the buffer in an amount of 10 to 25 pg/mL and the buffer comprises human tubal fluid medium or modified human tubal fluid medium.

13. A method of increasing a yield of motile spermatozoa in a plurality of spermatozoa after cryopreservation, comprising: providing a plurality of spermatozoa, contacting the plurality spermatozoa with hydroxyurea provided in a buffer, wherein the hydroxyurea is present in the buffer in an amount of 10 to 25 pg/mL and the buffer comprises human tubal fluid medium or modified human tubal fluid medium, subjecting the plurality of spermatozoa to cryopreservation, and thawing the plurality of spermatozoa.

14. The method of any of claims 11-13, further comprising incubating the spermatozoon in contact with the hydroxyurea for a period of from 1 minute to 24 hours.

15. The method of any of claims 11-13, wherein the spermatozoon is human spermatozoon or bovine spermatozoon.

16. The method of any of claims 11-13, wherein the contacted spermatozoon has an enhanced motility after being contacted with hydroxyurea and the enhanced motility is hyperactive motility or progressive motility.